PROJECT SUMMARY Children in the northern coast of Puerto Rico are exposed to a complex mix of environmental contaminants. The region has over 200 hazardous waste sites, including 16 active Superfund sites, resulting in contaminated water resources. Air pollution from refineries, power plants, motor vehicles, and large ships at ports is also very high, and made worse by Saharan dust storms crossing the Atlantic which have increased in frequency in recent decades. Children in highly polluted areas like Puerto Rico rarely experience any of these exposures in isolation, yet health effect studies associated with exposure to chemical mixtures are rare, with most research instead focusing on single chemicals. Significant challenges remain in understanding the complex risks that pollutant mixtures and their metabolites pose for human health. Novel approaches to examining the impacts of pollutant mixtures are critical to developing preventative strategies to reduce the burden of disease in children in Puerto Rico and the U.S. These challenges motivate this project, which will employ novel in vitro, high throughput screening (HTS) assays to yield new toxicity information needed to reveal the impacts of exposure to complex pollutant mixtures on neonatal and early childhood development in Puerto Rico. These HTS assays will specifically measure translational changes in oxidative stress, DNA damage and inflammation pathways occurring in response to pollutants and pollutant mixtures. Importantly, these pollutants and mixtures are drawn directly from environmental sources, such as water and air, and also from biological matrices like urine, providing information about exposures to chemical mixtures actually occurring in the real-world. Through this approach, we can provide pathway- and sample-specific fingerprints of pollutant exposures and their effects that can inform epidemiological studies, including CRECE Projects 1 and 3. These cross-project collaborations will enable CRECE to perform health effect analyses for pollutant mixtures identified as toxic by the HTS and to examine the association of exposure biomarkers with neonatal and early childhood health outcomes. We will use this approach to achieve four specific aims: (1) assess the toxicity of individual pollutants and pollutant mixtures found in tap water collected from 40 participants' homes; (2) quantify the toxicity associated with metal and organic carbon extracts from airborne fine particle (PM2.5) samples collected as part of Project 1; (3) analyze mixture toxicity effects of CECs, metals, and other chemicals and chemical metabolites in urine samples of 40 pregnant mothers, and (4) use toxicity measures from Aims 2 and 3 and pollutant concentrations measured in Projects 1 and 3 to identify pollutants most responsible for eliciting the observed pathway-specific toxicity impacts. The outcomes of this project will help to inform strategies to minimize health impacts from multiple exposures on Puerto Rico children, a highly exposed population that experiences disproportionate levels of childhood illness, as well as children in the U.S.